JP2023177269A - Method and system for assessing multi-level risks of antibiotic residues in water environment - Google Patents
Method and system for assessing multi-level risks of antibiotic residues in water environment Download PDFInfo
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- 230000003115 biocidal effect Effects 0.000 title claims abstract description 175
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 29
- 206010059866 Drug resistance Diseases 0.000 claims abstract description 58
- 230000000813 microbial effect Effects 0.000 claims abstract description 57
- 238000011156 evaluation Methods 0.000 claims abstract description 47
- 238000012544 monitoring process Methods 0.000 claims abstract description 47
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 39
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 39
- 238000012502 risk assessment Methods 0.000 claims abstract description 21
- KBPLFHHGFOOTCA-UHFFFAOYSA-N 1-Octanol Chemical compound CCCCCCCCO KBPLFHHGFOOTCA-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000012216 screening Methods 0.000 claims abstract description 14
- 238000005192 partition Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 58
- 230000007613 environmental effect Effects 0.000 claims description 47
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 239000012528 membrane Substances 0.000 claims description 20
- 239000008055 phosphate buffer solution Substances 0.000 claims description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 14
- 108020000946 Bacterial DNA Proteins 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 230000002688 persistence Effects 0.000 claims description 14
- 230000010365 information processing Effects 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 11
- 239000011324 bead Substances 0.000 claims description 10
- 239000011521 glass Substances 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 9
- 230000002085 persistent effect Effects 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000011148 porous material Substances 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000012137 tryptone Substances 0.000 claims description 5
- 230000032770 biofilm formation Effects 0.000 claims description 4
- 238000011835 investigation Methods 0.000 claims description 4
- 239000013049 sediment Substances 0.000 claims description 4
- 241001156739 Actinobacteria <phylum> Species 0.000 claims description 3
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 241000233866 Fungi Species 0.000 claims description 3
- IABBAGAOMDWOCW-UHFFFAOYSA-N Nicametate citrate Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O.CCN(CC)CCOC(=O)C1=CC=CN=C1 IABBAGAOMDWOCW-UHFFFAOYSA-N 0.000 claims description 3
- 230000036983 biotransformation Effects 0.000 claims description 3
- 239000006227 byproduct Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 239000012634 fragment Substances 0.000 description 6
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
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Abstract
Description
本発明は、環境保護の分野に関し、具体的に、水環境中の残留抗生物質に対するマルチレ
ベルのリスク評価方法および評価システムに関する。
The present invention relates to the field of environmental protection, and specifically relates to a multi-level risk assessment method and system for antibiotic residues in aquatic environments.
抗生物質は、細菌を殺すことによって感染症を治療するために使用されるが、広く存在す
る生物群としての細菌も、殺されるリスクを回避するために、様々な形で抗生物質に対す
る耐性を獲得可能であり、この耐性は「細菌薬品耐性」と呼ばれ、薬品耐性能力を獲得し
た細菌は「薬品耐性細菌」と呼ばれる。
生態リスクとは、生態系が生態系に脅威を与える生態系外のあらゆる要素にさらされる可
能性のことで、ある地域における化学物質の排出、人間活動、自然災害などによって生態
系やその構成要素に悪影響を及ぼし、生態系の構造や機能にダメージを与え、生態系の安
全や健康を脅かす可能性があることを指す。
Antibiotics are used to treat infections by killing bacteria, but bacteria as a widespread group of organisms also develop resistance to antibiotics in various ways to avoid the risk of being killed. This resistance is called "bacterial drug resistance," and bacteria that have acquired the ability to resist drugs are called "drug-resistant bacteria."
Ecological risk is the potential for an ecosystem to be exposed to any element outside the ecosystem that poses a threat to the ecosystem, such as the release of chemicals in a region, human activities, natural disasters, etc. refers to the possibility of having a negative impact on the environment, damaging the structure and function of the ecosystem, and threatening the safety and health of the ecosystem.
上記の問題に対して、本発明は、水環境中の残留抗生物質に対するマルチレベルのリスク
評価方法を提供する。
本発明の技術的解決策は以下の通りである。
水環境中の残留抗生物質に対するマルチレベルのリスク評価方法は、以下のステップを含
み、
Ｓ１、環境監視：
Ｓ１-１：目標流域における抗生物質の生産、使用および排出の背景調査を行い、生態リ
スク評価目標抗生物質の監視リストを確立し、目標抗生物質の環境監視を行い、前記目標
流域は河川流域、海域および水機能領域を含み、
Ｓ１-２：目標流域における目標抗生物質誘導体を監視し、目標抗生物質の副産物反応や
生物変換が発生した場合、目標抗生物質誘導体監視リストを確立し、目標抗生物質誘導体
の環境監視を行う必要もあり、
Ｓ２、抗生物質初期スクリーニング：ステップＳ１-１で得られた目標流域の目標抗生物
質およびステップＳ１-２で得られた目標流域の目標抗生物質誘導体を初期スクリーニン
グし、Ｓ２-１～Ｓ２-２のいずれかを満たす場合、ステップＳ３微生物薬品耐性評価に移
行して継続的に評価し、それ以外の場合、評価を終了し、
Ｓ２-１、オクタノール／水分配係数判定：目標抗生物質または目標抗生物質誘導体のオ
クタノール／水分配係数Ｋｏｗを求め、取得したオクタノール／水分配係数Ｋｏｗを１０
の底を持つ対数値とし、ｌｇ Ｋｏｗ≧３．５の場合、この目標抗生物質または目標抗生
物質誘導体が強脂溶性と判定し、ステップＳ３微生物薬品耐性評価に移行して継続的に評
価し、
Ｓ２-２、抗生物質環境濃度判定：目標抗生物質または目標抗生物質誘導体の環境濃度Ｍ
ＥＣを測定し、環境濃度ＭＥＣ≧１０の場合、この目標抗生物質または目標抗生物質誘導
体が連続的な入力源を有すると判定し、ステップＳ３微生物薬品耐性評価に移行して継続
的に評価する必要があり、
Ｓ３、微生物薬品耐性評価：薬品耐性リスク指数ＲＱＲを用いて目標流域中の微生物群集
の薬品耐性リスクを特徴付け、以下の式に示すように：
ＲＱＲ＝ＭＥＣ／ＰＮＥＣＲ、
式において、ＭＥＣはステップＳ２-２で測定された目標抗生物質または目標抗生物質誘
導体の環境濃度であり、ＰＮＥＣＲは目標流域中の微生物群集の予測された薬品耐性無効
濃度であり、以下の式に示すように：
ＰＮＥＣＲ＝ＭＩＣ／ＡＦ、
式において、ＭＩＣは目標流域中の微生物群集の最小発育阻止濃度であり、ＡＦは１０の
値を取る無次元評価要因であり、ＥＵＣＡＳＴデータベースから目標流域中の微生物群集
の最小発育阻止濃度ＭＩＣを決定し、目標流域中の微生物群集の予測された薬品耐性無効
濃度ＰＮＥＣＲを取得し、前記微生物群集は細菌、真菌または放線菌属を含み、
目標流域中の微生物群集の薬品耐性リスクレベルは、ＲＱＲ値の大きさに応じて、ＲＱＲ
＜０．０１でリスクなし、０．０１＜ＲＱＲ＜０．１で低リスク、０．１＜ＲＱＲ＜１で
中リスク、ＲＱＲ＞１で高リスクという４クラスに分けられ、ＲＱＲ＞０．１のとき、目
標流域中の微生物群集の薬品耐性リスクレベルが高いと見なされ、ステップＳ４高レベル
評価に移行して継続的に評価し、それ以外の場合、評価を終了し、
Ｓ４、高レベル評価：
Ｓ４-１、環境微生物抽出：５０重量部の目標流域水試料をフィルター膜で濾過し、前記
フィルター膜の孔径は０．２～０．３μｍであり、濾過後、フィルター膜を面積１５～２
８ｍｍ２の断片に切断し、断片を容器に入れ、断片体積の１．２～１．５倍のリン酸塩緩
衝溶液を加え、容器に断片体積の２．５～３倍のガラスビーズを添加し、ボルテックスで
１５～２０ｍｉｎ振とうした後、フィルター膜とガラスビーズを濾過・除去して、細菌溶
液上澄み液を得て用意し、
Ｓ４-２、抗生物質勾配濃度培養：ステップＳ４-１で得られた細菌溶液の上澄み液をそれ
ぞれ５～６個の減菌済コニカルフラスコに加え、各減菌済コニカルフラスコには２．５～
３重量部の細菌溶液上澄み液が含まれ、各減菌済コニカルフラスコに、堆積物のバイオフ
ィルム形成を模擬するためのスライドを３枚入れ、０．２～０．５重量部のＬＢ培地を加
え、５～６個の異なる減菌済コニカルフラスコに異なる濃度の目標抗生物質または目標抗
生物質誘導体を加え、５～６個の異なる減菌済コニカルフラスコ中の目標抗生物質または
目標抗生物質誘導体濃度はそれぞれ０、０．０１μｇ／Ｌ、０．１μｇ／Ｌ、１μｇ／Ｌ
、１０μｇ／Ｌ、１００μｇ／Ｌであり、２８～３３℃の室温条件下で１４日間振とう反
応させ、目標溶液を得、
Ｓ４-３、ＤＮＡ抽出：ステップＳ４-２で得られた目標溶液から目標溶液ＤＮＡを抽出す
る同時、減菌済コニカルフラスコ中のスライド上の細菌をリン酸塩緩衝溶液に収集して細
菌ＤＮＡを抽出し、
Ｓ４-４、耐性遺伝子測定：目標抗生物質または目標抗生物質誘導体に対応する耐性遺伝
子を測定し、目標溶液ＤＮＡまたは細菌ＤＮＡの存在量を測定して耐性遺伝子の相対存在
量の平均値Ｐを算出する同時に、ステップＳ４前の耐性遺伝子の相対存在量の平均値Ｐ０
を算出し、選択性係数Ｓを算出し、以下の式に示すように：
Ｓ＝Ｉｎ（Ｐ／Ｐ０）
Ｓ４-５、モデルフィッティング：抗生物質濃度Ｃを横座標とし、目標溶液ＤＮＡまたは
細菌ＤＮＡに対応する選択性係数Ｓを縦座標として、ｌｏｇｉｓｔｉｃモデルを用いてフ
ィッティングし、Ｓ＝０のとき、対応の抗生物質濃度ＣはＭＳＣ値であり、目標溶液ＤＮ
Ａまたは細菌ＤＮＡのＭＳＣ値を比較し、小さいＭＳＣ値を管理基準として選択し、この
ＭＳＣ値は、抗生物質濃度Ｃより高い抗生物質条件下で薬品耐性遺伝子を有する微生物が
濃縮され、その結果、微生物群集中の薬品耐性遺伝子の相対存在量が増加する。
本発明の一側面として、前記ステップＳ３は以下のステップをさらに含み、環境残留性評
価：データベースクエリーとモデル予測方法を組み合わせて目標抗生物質または目標抗生
物質誘導体の半減期ｔ１／２を求め、環境残留性レベルは半減期ｔ１／２値の大きさに応
じて、ｔ１／２＜６０ｄで非残留性、６０ｄ＜ｔ１／２＜１８０ｄで残留性、ｔ１／２＞
１８０ｄで高残留性という３クラスに分けられ、ｔ１／２＞６０の場合、目標抗生物質ま
たは目標抗生物質誘導体が環境残留性を有すると判定し、すなわち、目標流域中の微生物
群集の薬品耐性リスクＲＱＲの値にかかわらず、ステップＳ４に移行して継続的に評価す
る必要がある。環境残留性と微生物薬品耐性を組み合わせることにより、目標抗生物質ま
たは目標抗生物質誘導体が残留性と薬品耐性がある場合には高リスク評価を行い、残留性
がなく薬品耐性がある場合には適時に高レベルリスク評価を行うという、より正確な評価
方法を実現することができる。
本発明の一側面として、前記ステップＳ４-１とＳ４-３中のリン酸塩緩衝溶液では、Ｎａ
Ｃｌ溶液の質量濃度が１０ｇ／Ｌ、ＫＣｌ溶液の質量濃度が０．２５ｇ／Ｌ、Ｎａ２ＨＰ
Ｏ４溶液の質量濃度が１．６ｇ／Ｌ、ＫＨ２ＰＯ４溶液の質量濃度が０．３ｇ／Ｌであり
、残りは水であり、リン酸塩緩衝溶液のｐＨは７．４である。このリン酸塩緩衝溶液は細
菌と相性が良い。
本発明の一側面として、前記ステップＳ４-１中のＬＢ培地では、トリプトンの質量濃度
が１０ｇ／Ｌ、酵母粉末の質量濃度が５ｇ／Ｌ、塩化ナトリウムの質量濃度が１０ｇ／Ｌ
であり、残りは水である。このＬＢ培地は細菌の培養に有効である。
本発明の一側面として、前記ステップＳ４-４では２～２０種類の耐性遺伝子を取る。
本発明は、上記方法で使用される、水環境中の残留抗生物質に対するマルチレベルのリス
ク評価システムをさらに提供し、この評価システムは、
目標流域内の目標抗生物質および目標抗生物質誘導体の種類および含有量を監視するため
に使用され、まず情報処理装置において目標流域内の目標抗生物質監視リスト、目標抗生
物質誘導体監視リストを作成し、その後目標抗生物質および目標抗生物質誘導体の監視を
実行する環境監視システムと、
情報処理装置上で実際に検出した目標抗生物質、目標抗生物質誘導体と、環境監視システ
ムにより建築された目標抗生物質監視リスト、目標抗生物質誘導体監視リストとを比較し
、実際に検出した目標抗生物質、目標抗生物質誘導体が環境監視システムにより建築され
た目標抗生物質監視リスト、目標抗生物質誘導体監視リストに属するかどうかを判定する
初期スクリーニングシステムと、
情報処理装置上で、前記初期スクリーニングシステムの判定結果がＹｅｓである目標抗生
物質、目標抗生物質誘導体に対して微生物薬品耐性評価を行う微生物薬品耐性評価システ
ムと、
情報処理装置上で微生物薬品耐性リスクレベルの高い目標抗生物質、目標抗生物質誘導体
をさらに評価して、制限または禁止する必要のある生態学的リスクの高い抗生物質種類を
特定する高レベル評価システムと、を備える。
To address the above problems, the present invention provides a multi-level risk assessment method for antibiotic residues in aquatic environments.
The technical solution of the present invention is as follows.
The multi-level risk assessment method for antibiotic residues in the water environment includes the following steps:
S1, environmental monitoring:
S1-1: Conduct a background investigation on the production, use and discharge of antibiotics in the target watershed, establish an ecological risk assessment monitoring list for the target antibiotics, perform environmental monitoring of the target antibiotics, and the target watershed is a river basin, including marine areas and water functional areas;
S1-2: Monitor the target antibiotic derivatives in the target watershed, and if by-product reactions or biotransformations of the target antibiotics occur, it is also necessary to establish a target antibiotic derivative monitoring list and conduct environmental monitoring of the target antibiotic derivatives. can be,
S2, Initial screening of antibiotics: Initial screening of the target antibiotic in the target area obtained in step S1-1 and the target antibiotic derivative in the target area obtained in step S1-2, and If any of the above is satisfied, proceed to step S3 Microbial drug resistance evaluation and continuously evaluate; otherwise, end the evaluation,
S2-1, Determination of octanol/water partition coefficient: determine the octanol/water partition coefficient Kow of the target antibiotic or target antibiotic derivative, and set the obtained octanol/water partition coefficient Kow to 10
If lg Kow≧3.5, the target antibiotic or target antibiotic derivative is determined to be strongly lipophilic, and the process proceeds to step S3, microbial drug resistance evaluation, where it is continuously evaluated.
S2-2, Antibiotic environmental concentration determination: Environmental concentration M of target antibiotic or target antibiotic derivative
EC is measured, and if the environmental concentration MEC≧10, it is determined that this target antibiotic or target antibiotic derivative has a continuous input source, and it is necessary to proceed to step S3 microbial drug resistance evaluation and continuously evaluate. There is,
S3. Microbial drug resistance evaluation: The drug resistance risk index RQ R is used to characterize the drug resistance risk of the microbial community in the target watershed, as shown in the following formula:
RQ R =MEC/PNEC R ,
In the formula, MEC is the environmental concentration of the target antibiotic or target antibiotic derivative measured in step S2-2, PNEC R is the predicted drug resistance null concentration of the microbial community in the target watershed, and the following formula As shown:
PNEC R = MIC/AF,
In the formula, MIC is the minimum inhibitory concentration of the microbial community in the target watershed, AF is a dimensionless evaluation factor that takes a value of 10, and the minimum inhibitory concentration MIC of the microbial community in the target watershed is determined from the EUCAST database. obtain a predicted drug resistance null concentration PNECR of a microbial community in a target watershed, the microbial community comprising bacteria, fungi, or the genus Actinobacteria;
The drug resistance risk level of the microbial community in the target watershed is determined by the RQ R value.
RQ R is divided into four classes: no risk if <0.01, low risk if 0.01<RQ R <0.1, medium risk if 0.1<RQ R <1, and high risk if RQ R >1. >0.1, the drug resistance risk level of the microbial community in the target watershed is considered to be high, and the process moves to step S4 high level evaluation to continuously evaluate; otherwise, the evaluation is terminated;
S4, high level evaluation:
S4-1, Extraction of environmental microorganisms: 50 parts by weight of the target watershed water sample is filtered with a filter membrane, the pore size of the filter membrane is 0.2~0.3μm, and after filtration, the filter membrane has an area of 15~2μm.
Cut into 8 mm 2 pieces, place the pieces in a container, add 1.2 to 1.5 times the volume of the pieces in phosphate buffer solution, and add 2.5 to 3 times the volume of glass beads in the container. After shaking with a vortex for 15 to 20 minutes, filter and remove the filter membrane and glass beads to obtain and prepare a bacterial solution supernatant.
S4-2, Antibiotic gradient concentration culture: Add the supernatant of the bacterial solution obtained in step S4-1 to 5 to 6 sterilized conical flasks, and add 2.5 to
Each sterilized conical flask contained 3 parts by weight of bacterial solution supernatant, 3 slides to simulate sediment biofilm formation, and 0.2 to 0.5 parts by weight of LB medium. In addition, add different concentrations of the target antibiotic or target antibiotic derivative to 5 to 6 different sterilized conical flasks to determine the target antibiotic or target antibiotic derivative concentration in 5 to 6 different sterilized conical flasks. are 0, 0.01 μg/L, 0.1 μg/L, and 1 μg/L, respectively.
.
S4-3, DNA extraction: At the same time as extracting the target solution DNA from the target solution obtained in step S4-2, collect the bacteria on the slide in a sterilized conical flask into a phosphate buffer solution to extract bacterial DNA. extract,
S4-4, Resistance gene measurement: Measure the resistance gene corresponding to the target antibiotic or target antibiotic derivative, measure the abundance of target solution DNA or bacterial DNA, and calculate the average value P of the relative abundance of the resistance gene. At the same time, the average value P 0 of the relative abundance of resistance genes before step S4
and calculate the selectivity coefficient S, as shown in the following formula:
S=In(P/P 0 )
S4-5, model fitting: fitting using a logistic model with the antibiotic concentration C as the abscissa and the selectivity coefficient S corresponding to the target solution DNA or bacterial DNA as the ordinate, and when S = 0, the corresponding The antibiotic concentration C is the MSC value and the target solution DN
The MSC value of A or bacterial DNA is compared, and the smaller MSC value is selected as the control standard, and this MSC value indicates that microorganisms with drug resistance genes are enriched under antibiotic conditions higher than the antibiotic concentration C, and as a result, The relative abundance of drug resistance genes in the microbial community increases.
In one aspect of the present invention, step S3 further includes the following steps: environmental persistence assessment: determining the half-life t 1/2 of the target antibiotic or target antibiotic derivative by combining a database query and a model prediction method; The environmental persistence level depends on the half-life t 1/2 value: non-persistent when t 1/2 <60 d, persistent when 60 d < t 1/2 <180 d, and persistent when t 1/2 >
If t 1/2 > 60, the target antibiotic or target antibiotic derivative is determined to have environmental persistence, that is, the drug resistance of the microbial community in the target watershed is determined to be high. Regardless of the value of risk RQR , it is necessary to proceed to step S4 and continuously evaluate. By combining environmental persistence and microbial drug resistance, a high-risk assessment can be made if the target antibiotic or target antibiotic derivative is persistent and drug-resistant, and a timely assessment if the target antibiotic or target antibiotic derivative is persistent and drug-resistant. It is possible to realize a more accurate evaluation method of performing a high-level risk assessment.
As one aspect of the present invention, in the phosphate buffer solution in steps S4-1 and S4-3, Na
The mass concentration of Cl solution is 10 g/L, the mass concentration of KCl solution is 0.25 g/L, Na 2 HP
The mass concentration of the O 4 solution is 1.6 g/L, the mass concentration of the KH 2 PO 4 solution is 0.3 g/L, the remainder is water, and the pH of the phosphate buffer solution is 7.4. This phosphate buffer solution is compatible with bacteria.
As one aspect of the present invention, in the LB medium in step S4-1, the mass concentration of tryptone is 10 g/L, the mass concentration of yeast powder is 5 g/L, and the mass concentration of sodium chloride is 10 g/L.
and the rest is water. This LB medium is effective for culturing bacteria.
As one aspect of the present invention, in step S4-4, 2 to 20 types of resistance genes are selected.
The present invention further provides a multi-level risk assessment system for antibiotic residues in a water environment for use in the above method, which assessment system comprises:
It is used to monitor the types and contents of target antibiotics and target antibiotic derivatives in the target watershed, and first, a target antibiotic monitoring list and a target antibiotic derivative monitoring list in the target watershed are created in the information processing device, an environmental monitoring system that subsequently performs monitoring of the target antibiotic and target antibiotic derivative;
The target antibiotics and target antibiotic derivatives actually detected on the information processing device are compared with the target antibiotic monitoring list and target antibiotic derivative monitoring list constructed by the environmental monitoring system, and the target antibiotics actually detected are determined. , an initial screening system for determining whether the target antibiotic derivative belongs to a target antibiotic derivative monitoring list, a target antibiotic derivative monitoring list established by an environmental monitoring system;
a microbial drug resistance evaluation system that performs microbial drug resistance evaluation on a target antibiotic or target antibiotic derivative for which the determination result of the initial screening system is Yes on an information processing device;
A high-level evaluation system that further evaluates target antibiotics and target antibiotic derivatives with a high risk level of microbial drug resistance on an information processing device to identify types of antibiotics with a high ecological risk that need to be restricted or prohibited. , is provided.
本発明は以下の有益な効果を有する。
（１）本発明の水環境中の残留抗生物質に対するマルチレベルのリスク評価方法は、リス
クを有する水環境目標抗生物質または目標抗生物質誘導体に対してカスケード評価を行い
、まず目標抗生物質または目標抗生物質誘導体を初期スクリーニングし、条件を満たせば
微生物薬品耐性評価に移行して次の評価を行い、薬品耐性の相違について、継続的に高レ
ベル評価およびリスク評価を行うか、または評価を終了し、最終的にＭＳＣ値を得、これ
は薬品耐性遺伝子を持つ微生物が対応の抗生物質濃度Ｃよりも高い抗生物質条件下で濃縮
され、微生物群集における薬品耐性遺伝子の相対存在量が増加することを示す。
（２）本発明の水環境中の残留抗生物質に対するマルチレベルのリスク評価方法は、環境
残留性と微生物薬品耐性を組み合わせることにより、目標抗生物質または目標抗生物質誘
導体が残留性と薬品耐性がある場合には高リスク評価を行い、残留性がなく薬品耐性があ
る場合には適時に高レベルリスク評価を行うという、より正確な評価方法を実現すること
ができる。
（３）本発明の水環境中の残留抗生物質に対するマルチレベルのリスク評価方法は、実験
方法により、ＭＳＣ値を正確に測定し、抗生物質濃度Ｃを横座標、目標溶液ＤＮＡまたは
細菌ＤＮＡに対応する選択性係数Ｓを縦座標とし、ｌｏｇｉｓｔｉｃモデルでフィッティ
ングして目標溶液ＤＮＡまたは細菌ＤＮＡのＭＳＣ値を得、その小さいＭＳＣ値を管理基
準とする。
（４）本発明の水環境中の残留抗生物質に対するマルチレベルのリスク評価方法で得られ
た評価結果は、水環境の連続監視、発生源分析および発生源管理において重要な役割を果
たし、評価結果によると、点源による汚染について調査・処罰、期限是正などの行政措置
を適用でき、面源による汚染について生態学的リスクの高い抗生物質種の使用を関連地域
で制限または禁止することができる。
The present invention has the following beneficial effects.
(1) The multi-level risk assessment method for residual antibiotics in the water environment of the present invention performs a cascade evaluation on target antibiotics or target antibiotic derivatives in the water environment that pose a risk. Perform initial screening of substance derivatives, and if conditions are met, proceed to microbial drug resistance evaluation and perform the next evaluation, and continue high-level evaluation and risk assessment for differences in drug resistance, or terminate the evaluation. We finally obtained the MSC value, which indicates that microorganisms with drug resistance genes are enriched under antibiotic conditions higher than the corresponding antibiotic concentration C, increasing the relative abundance of drug resistance genes in the microbial community. .
(2) The multi-level risk assessment method for residual antibiotics in the water environment of the present invention combines environmental persistence and microbial drug resistance to ensure that the target antibiotic or target antibiotic derivative has persistence and drug resistance. A more accurate evaluation method can be realized in which a high-level risk assessment is performed in a timely manner when there is no persistence and there is drug resistance.
(3) The multi-level risk assessment method for residual antibiotics in the water environment of the present invention uses an experimental method to accurately measure the MSC value, and the antibiotic concentration C is the abscissa, corresponding to the target solution DNA or bacterial DNA. The selectivity coefficient S is taken as the ordinate, and the MSC value of the target solution DNA or bacterial DNA is obtained by fitting with a logistic model, and the smaller MSC value is used as the control standard.
(4) The evaluation results obtained by the multilevel risk assessment method for antibiotic residues in the water environment of the present invention play an important role in continuous monitoring, source analysis, and source control of the water environment, and the evaluation results According to the Ministry of Health, Labor and Welfare, administrative measures such as investigation, punishment, and correction of deadlines can be applied to point source pollution, and the use of antibiotic species with high ecological risks can be restricted or prohibited in relevant areas for point source pollution.
実施例１
水環境中の残留抗生物質に対するマルチレベルのリスク評価方法は、以下のステップを含
み、
Ｓ１、環境監視：
Ｓ１-１：目標流域における抗生物質の生産、使用および排出の背景調査を行い、生態リ
スク評価目標抗生物質の監視リストを確立し、目標抗生物質の環境監視を行い、前記目標
流域は河川流域であり、
Ｓ１-２：目標流域における目標抗生物質誘導体を監視し、目標抗生物質の副産物反応や
生物変換が発生した場合、目標抗生物質誘導体監視リストを確立し、目標抗生物質誘導体
の環境監視を行う必要もあり、
Ｓ２、抗生物質初期スクリーニング：ステップＳ１-１で得られた目標流域の目標抗生物
質およびステップＳ１-２で得られた目標流域の目標抗生物質誘導体を初期スクリーニン
グし、Ｓ２-１～Ｓ２-２のいずれかを満たす場合、ステップＳ３微生物薬品耐性評価に移
行して継続的に評価し、それ以外の場合、評価を終了し、
Ｓ２-１、オクタノール／水分配係数判定：目標抗生物質または目標抗生物質誘導体のオ
クタノール／水分配係数Ｋｏｗを求め、取得したオクタノール／水分配係数Ｋｏｗを１０
の底を持つ対数値とし、ｌｇ Ｋｏｗ≧３．５の場合、この目標抗生物質または目標抗生
物質誘導体が強脂溶性と判定し、ステップＳ３微生物薬品耐性評価に移行して継続的に評
価し、一部の抗生物質のｌｏｇ ｋｏｗ 値は表２に示され、
Ｓ２-２、抗生物質環境濃度判定：目標抗生物質または目標抗生物質誘導体の環境濃度Ｍ
ＥＣを測定し、環境濃度ＭＥＣ≧１０の場合、この目標抗生物質または目標抗生物質誘導
体が連続的な入力源を有すると判定し、ステップＳ３微生物薬品耐性評価に移行して継続
的に評価する必要があり、
Ｓ３、微生物薬品耐性評価：薬品耐性リスク指数ＲＱＲを用いて目標流域中の微生物群集
の薬品耐性リスクを特徴付け、微生物群集は細菌であり、以下の式に示すように：
ＲＱＲ＝ＭＥＣ／ＰＮＥＣＲ、
式において、ＭＥＣはステップＳ２-２で測定された目標抗生物質または目標抗生物質誘
導体の環境濃度であり、ＰＮＥＣＲは目標流域中の微生物群集の予測された薬品耐性無効
濃度であり、以下の式に示すように：
ＰＮＥＣＲ＝ＭＩＣ／ＡＦ、
式において、ＭＩＣは目標流域中の微生物群集の最小発育阻止濃度であり、ＡＦは１０の
値を取る無次元評価要因であり、ＥＵＣＡＳＴデータベースから目標流域中の微生物群集
の最小発育阻止濃度ＭＩＣを決定し、目標流域中の微生物群集の予測された薬品耐性無効
濃度ＰＮＥＣＲを取得し、一部の抗生物質の最小発育阻止濃度（ＭＩＣ）と微生物薬品耐
性予測無効濃度（ＰＮＥＣｒ）は表３に示され、
目標流域中の微生物群集の薬品耐性リスクレベルは、ＲＱＲ値の大きさに応じて、ＲＱＲ
＜０．０１でリスクなし、０．０１＜ＲＱＲ＜０．１で低リスク、０．１＜ＲＱＲ＜１で
中リスク、ＲＱＲ＞１で高リスクという４クラスに分けられ、ＲＱＲ＞０．１のとき、目
標流域中の微生物群集の薬品耐性リスクレベルが高いと見なされ、ステップＳ４高レベル
評価に移行して継続的に評価し、それ以外の場合、評価を終了し、
Ｓ４、高レベル評価：
Ｓ４-１、環境微生物抽出：５０重量部の目標流域水試料をフィルター膜で濾過し、フィ
ルター膜の孔径は０．２５μｍであり、濾過後、フィルター膜を面積２０ｍｍ２の断片に
切断し、断片を容器に入れ、断片体積の１．３倍のリン酸塩緩衝溶液を加え、リン酸塩緩
衝溶液では、ＮａＣｌ溶液の質量濃度が１０ｇ／Ｌ、ＫＣｌ溶液の質量濃度が０．２５ｇ
／Ｌ、Ｎａ２ＨＰＯ４溶液の質量濃度が１．６ｇ／Ｌ、ＫＨ２ＰＯ４溶液の質量濃度が０
．３ｇ／Ｌであり、残りは水であり、リン酸塩緩衝溶液のｐＨは７．４であり、さらに、
容器に断片体積の２．８倍のガラスビーズを添加し、ボルテックスで１８ｍｉｎ振とうし
た後、フィルター膜とガラスビーズを濾過・除去して、細菌溶液上澄み液を得て用意し、
Ｓ４-２、抗生物質勾配濃度培養：ステップＳ４-１で得られた細菌溶液上澄み液をそれぞ
れ５個の減菌済コニカルフラスコに加え、各減菌済コニカルフラスコには２．７重量部の
細菌溶液上澄み液が含まれ、各減菌済コニカルフラスコに、堆積物のバイオフィルム形成
を模擬するためのスライドを３枚入れ、０．４重量部のＬＢ培地を加え、ＬＢ培地では、
トリプトンの質量濃度が１０ｇ／Ｌ、酵母粉末の質量濃度が５ｇ／Ｌ、塩化ナトリウムの
質量濃度が１０ｇ／Ｌであり、残りは水であり、さらに５個の異なる減菌済コニカルフラ
スコに異なる濃度の目標抗生物質または目標抗生物質誘導体を加え、５個の異なる減菌済
コニカルフラスコ中の目標抗生物質または目標抗生物質誘導体の濃度はそれぞれ０．０１
μｇ／Ｌ、０．１μｇ／Ｌ、１μｇ／Ｌ、１０μｇ／Ｌ、１００μｇ／Ｌであり、３０℃
の室温条件下で１４日間振とう反応させて目標溶液を得、
Ｓ４-３、ＤＮＡ抽出：ステップＳ４-２で得られた目標溶液から目標溶液ＤＮＡを抽出す
る同時に、減菌済コニカルフラスコ中のスライド上の細菌をリン酸塩緩衝溶液に収集して
細菌ＤＮＡを抽出し、
Ｓ４-４、耐性遺伝子測定：目標抗生物質または目標抗生物質誘導体に対応する耐性遺伝
子を測定し、耐性遺伝子は２～２０種を取り、２０種未満の場合最大値を取り、抗生物質
に対応する耐性遺伝子種類は表４に示され、目標溶液ＤＮＡまたは細菌ＤＮＡの存在量を
測定して耐性遺伝子の相対存在量の平均値Ｐを算出する同時に、ステップＳ４以前の耐性
遺伝子の相対存在量の平均値Ｐ０も算出し、以下の式に示すように選択性係数Ｓを算出し
：
Ｓ＝Ｉｎ（Ｐ／Ｐ０）
Ｓ４-５、モデルフィッティング：抗生物質濃度Ｃを横座標とし、目標溶液ＤＮＡまたは
細菌ＤＮＡに対応する選択性係数Ｓを縦座標として、ｌｏｇｉｓｔｉｃモデルを用いてフ
ィッティングし、Ｓ＝０のとき、対応の抗生物質濃度ＣはＭＳＣ値であり、目標溶液ＤＮ
Ａまたは細菌ＤＮＡのＭＳＣ値を比較し、小さいＭＳＣ値を管理基準として選択し、この
ＭＳＣ値は、抗生物質濃度Ｃより高い抗生物質条件下で薬品耐性遺伝子を有する微生物が
濃縮され、その結果、微生物群集中の薬品耐性遺伝子の相対存在量が増加する。
Example 1
The multi-level risk assessment method for antibiotic residues in the water environment includes the following steps:
S1, environmental monitoring:
S1-1: Conduct a background investigation on the production, use and discharge of antibiotics in the target watershed, establish a monitoring list of target antibiotics for ecological risk assessment, perform environmental monitoring of target antibiotics, and ensure that the target watershed is a river basin. can be,
S1-2: Monitor the target antibiotic derivatives in the target watershed, and if by-product reactions or biotransformations of the target antibiotics occur, it is also necessary to establish a target antibiotic derivative monitoring list and conduct environmental monitoring of the target antibiotic derivatives. can be,
S2, Initial screening of antibiotics: Initial screening of the target antibiotic in the target area obtained in step S1-1 and the target antibiotic derivative in the target area obtained in step S1-2, and If any of the above is satisfied, proceed to step S3 Microbial drug resistance evaluation and continuously evaluate; otherwise, end the evaluation,
S2-1, Determination of octanol/water partition coefficient: determine the octanol/water partition coefficient Kow of the target antibiotic or target antibiotic derivative, and set the obtained octanol/water partition coefficient Kow to 10
If lg Kow≧3.5, the target antibiotic or target antibiotic derivative is determined to be strongly lipophilic, and the process proceeds to step S3, microbial drug resistance evaluation, where it is continuously evaluated. The log kow values of some antibiotics are shown in Table 2,
S2-2, Antibiotic environmental concentration determination: Environmental concentration M of target antibiotic or target antibiotic derivative
EC is measured, and if the environmental concentration MEC≧10, it is determined that this target antibiotic or target antibiotic derivative has a continuous input source, and it is necessary to proceed to step S3 microbial drug resistance evaluation and continuously evaluate. There is,
S3, Microbial drug resistance evaluation: The drug resistance risk index RQR is used to characterize the drug resistance risk of the microbial community in the target watershed, where the microbial community is bacteria, as shown in the following formula:
RQ R =MEC/PNEC R ,
In the formula, MEC is the environmental concentration of the target antibiotic or target antibiotic derivative measured in step S2-2, PNEC R is the predicted drug resistance null concentration of the microbial community in the target watershed, and the following formula As shown:
PNEC R = MIC/AF,
In the formula, MIC is the minimum inhibitory concentration of the microbial community in the target watershed, AF is a dimensionless evaluation factor that takes a value of 10, and the minimum inhibitory concentration MIC of the microbial community in the target watershed is determined from the EUCAST database. The predicted antimicrobial resistance concentrations PNECR of the microbial community in the target watershed were obtained, and the minimum inhibitory concentrations (MICs) and predicted antimicrobial drug resistance concentrations (PNECr) of some antibiotics are shown in Table 3. ,
The drug resistance risk level of the microbial community in the target watershed is determined by the RQR value depending on the RQR value .
RQ R is divided into four classes: no risk if <0.01, low risk if 0.01<RQ R <0.1, medium risk if 0.1<RQ R <1, and high risk if RQ R >1. >0.1, the drug resistance risk level of the microbial community in the target watershed is considered to be high, and the process moves to step S4 high level evaluation to continuously evaluate; otherwise, the evaluation is terminated;
S4, high level evaluation:
S4-1, Environmental microorganism extraction: 50 parts by weight of target watershed water sample is filtered through a filter membrane, the pore size of the filter membrane is 0.25 μm, and after filtration, the filter membrane is cut into pieces with an area of 20 mm2 , and the pieces are into a container, add phosphate buffer solution 1.3 times the volume of the fragment, and in the phosphate buffer solution, the mass concentration of NaCl solution is 10 g/L and the mass concentration of KCl solution is 0.25 g/L.
/L, the mass concentration of Na 2 HPO 4 solution is 1.6 g/L, the mass concentration of KH 2 PO 4 solution is 0
．． 3 g/L, the remainder is water, the pH of the phosphate buffer solution is 7.4, and further,
Add glass beads 2.8 times the fragment volume to the container, shake with vortex for 18 min, filter and remove the filter membrane and glass beads to obtain and prepare a bacterial solution supernatant,
S4-2, Antibiotic gradient concentration culture: Add the bacterial solution supernatant obtained in step S4-1 to five sterilized conical flasks, and add 2.7 parts by weight of bacteria to each sterilized conical flask. Three slides were placed in each sterilized conical flask containing the solution supernatant to simulate sediment biofilm formation, and 0.4 parts by weight of LB medium was added.
The mass concentration of tryptone is 10 g/L, the mass concentration of yeast powder is 5 g/L, the mass concentration of sodium chloride is 10 g/L, the remainder is water, and five different sterilized conical flasks have different concentrations. of target antibiotic or target antibiotic derivative were added, and the concentration of target antibiotic or target antibiotic derivative in five different sterile conical flasks was 0.01, respectively.
μg/L, 0.1 μg/L, 1 μg/L, 10 μg/L, 100 μg/L, and at 30°C
The target solution was obtained by a shaking reaction for 14 days at room temperature.
S4-3, DNA extraction: At the same time, extract the target solution DNA from the target solution obtained in step S4-2, and collect the bacteria on the slide in a sterilized conical flask into a phosphate buffer solution to extract bacterial DNA. extract,
S4-4, Resistance gene measurement: Measure the resistance gene corresponding to the target antibiotic or target antibiotic derivative, select 2 to 20 resistance genes, and if there are less than 20 types, take the maximum value and determine the resistance gene corresponding to the antibiotic. The types of resistance genes are shown in Table 4, and the abundance of target solution DNA or bacterial DNA is measured to calculate the average value P of the relative abundance of resistance genes.At the same time, the average relative abundance of resistance genes before step S4 is calculated. The value P 0 is also calculated and the selectivity coefficient S is calculated as shown in the following formula:
S=In(P/P 0 )
S4-5, model fitting: fitting using a logistic model with the antibiotic concentration C as the abscissa and the selectivity coefficient S corresponding to the target solution DNA or bacterial DNA as the ordinate, and when S = 0, the corresponding The antibiotic concentration C is the MSC value and the target solution DN
The MSC value of A or bacterial DNA is compared, and the smaller MSC value is selected as the control standard, and this MSC value indicates that microorganisms with drug resistance genes are enriched under antibiotic conditions higher than the antibiotic concentration C, and as a result, The relative abundance of drug resistance genes in the microbial community increases.
実施例２
本実施例は、上記実施例１で用いた水環境中の残留抗生物質に対するマルチレベルのリス
ク評価システムであり、このシステムは、
目標流域内の目標抗生物質および目標抗生物質誘導体の種類および含有量を監視するため
に使用され、まず情報処理装置において目標流域内の目標抗生物質監視リスト、目標抗生
物質誘導体監視リストを作成し、その後目標抗生物質および目標抗生物質誘導体の監視を
実行する環境監視システムと、
情報処理装置上で実際に検出した目標抗生物質、目標抗生物質誘導体と、環境監視システ
ムにより建築された目標抗生物質監視リスト、目標抗生物質誘導体監視リストとを比較し
、実際に検出した目標抗生物質、目標抗生物質誘導体が環境監視システムにより建築され
た目標抗生物質監視リスト、目標抗生物質誘導体監視リストに属するかどうかを判定する
初期スクリーニングシステムと、
情報処理装置上で、前記初期スクリーニングシステムの判定結果がＹｅｓである目標抗生
物質、目標抗生物質誘導体に対して微生物薬品耐性評価を行う微生物薬品耐性評価システ
ムと、
情報処理装置上で微生物薬品耐性リスクレベルの高い目標抗生物質、目標抗生物質誘導体
をさらに評価して、制限または禁止する必要のある生態学的リスクの高い抗生物質種類を
特定する高レベル評価システムと、を備える。
Example 2
This example is a multi-level risk assessment system for residual antibiotics in the water environment used in Example 1 above.
It is used to monitor the types and contents of target antibiotics and target antibiotic derivatives in the target watershed, and first, a target antibiotic monitoring list and a target antibiotic derivative monitoring list in the target watershed are created in the information processing device, an environmental monitoring system that subsequently performs monitoring of the target antibiotic and target antibiotic derivative;
The target antibiotics and target antibiotic derivatives actually detected on the information processing device are compared with the target antibiotic monitoring list and target antibiotic derivative monitoring list constructed by the environmental monitoring system, and the target antibiotics actually detected are determined. , an initial screening system for determining whether the target antibiotic derivative belongs to a target antibiotic derivative monitoring list, a target antibiotic derivative monitoring list established by an environmental monitoring system;
a microbial drug resistance evaluation system that performs microbial drug resistance evaluation on a target antibiotic or target antibiotic derivative for which the determination result of the initial screening system is Yes on an information processing device;
A high-level evaluation system that further evaluates target antibiotics and target antibiotic derivatives with a high risk level of microbial drug resistance on an information processing device to identify types of antibiotics with a high ecological risk that need to be restricted or prohibited. , is provided.
実施例３
本実施例は、実施例１とは以下の点で異なり：
目標流域は海域である。
Example 3
This example differs from Example 1 in the following points:
The target basin is the sea area.
実施例４
本実施例は、実施例１とは以下の点で異なり：
目標流域は水機能領域、例えば溜池である。
Example 4
This example differs from Example 1 in the following points:
The target watershed is a water functional area, such as a reservoir.
実施例５
本実施例は、実施例１とは以下の点で異なり：
ステップＳ３中の微生物群集は真菌である。
Example 5
This example differs from Example 1 in the following points:
The microbial community in step S3 is fungi.
実施例６
本実施例は、実施例１とは以下の点で異なり：
ステップＳ３中の微生物群集は放線菌属である。
Example 6
This example differs from Example 1 in the following points:
The microbial community in step S3 is of the genus Actinobacteria.
実施例７
本実施例は、実施例１とは以下の点で異なり：
ステップＳ３は以下のステップをさらに含み、環境残留性評価：データベースクエリーと
モデル予測方法を組み合わせて目標抗生物質または目標抗生物質誘導体の半減期ｔ１／２
を求め、環境残留性レベルは半減期ｔ１／２値の大きさに応じて、ｔ１／２＜６０ｄで非
残留性、６０ｄ＜ｔ１／２＜１８０ｄで残留性、ｔ１／２＞１８０ｄで高残留性という３
クラスに分けられ、ｔ１／２＞６０の場合、目標抗生物質または目標抗生物質誘導体が環
境残留性を有すると判定し、すなわち、目標流域中の微生物群集の薬品耐性リスクＲＱＲ
の値にかかわらず、表５に示すように、ステップＳ４に移行して継続的に評価する必要が
ある。
Example 7
This example differs from Example 1 in the following points:
Step S3 further includes the following steps: environmental persistence assessment: combining database queries and model prediction methods to determine the half-life t1 /2 of the target antibiotic or target antibiotic derivative;
The environmental persistence level is determined according to the half-life t 1/2 value: non-persistent when t 1/2 <60 d, persistent when 60 d < t 1/2 <180 d, and persistent when t 1/2 > 3 with high persistence at 180d
If t 1/2 > 60, it is determined that the target antibiotic or target antibiotic derivative has environmental persistence, that is, the drug resistance risk of the microbial community in the target watershed RQ R
Regardless of the value of , as shown in Table 5, it is necessary to proceed to step S4 and continuously evaluate.
実施例８
本実施例は、実施例１とは以下の点で異なり：
Ｓ４-１、環境微生物抽出：５０重量部の目標流域水試料をフィルター膜で濾過し、フィ
ルター膜の孔径は０．２μｍであり、濾過後、フィルター膜を面積１５ｍｍ２の断片に切
断し、断片を容器に入れ、断片体積の１．２倍のリン酸塩緩衝溶液を加え、リン酸塩緩衝
溶液では、ＮａＣｌ溶液の質量濃度が１０ｇ／Ｌ、ＫＣｌ溶液の質量濃度が０．２５ｇ／
Ｌ、Ｎａ２ＨＰＯ４溶液の質量濃度が１．６ｇ／Ｌ、ＫＨ２ＰＯ４溶液の質量濃度が０．
３ｇ／Ｌであり、残りは水であり、リン酸塩緩衝溶液のｐＨは７．４であり、さらに容器
に断片体積の２．５倍のガラスビーズを添加し、ボルテックスで１５ｍｉｎ振とうした後
、フィルター膜とガラスビーズを濾過・除去して細菌溶液上澄み液を得て用意し、
Ｓ４-２、抗生物質勾配濃度培養：ステップＳ４-１で得られた細菌溶液上澄み液をそれぞ
れ５個の減菌済コニカルフラスコに加え、各減菌済コニカルフラスコには２．５重量部の
細菌溶液上澄み液が含まれ、各減菌済コニカルフラスコに、堆積物のバイオフィルム形成
を模擬するためのスライドを３枚入れ、０．２重量部のＬＢ培地を加え、ＬＢ培地では、
トリプトンの質量濃度が１０ｇ／Ｌ、酵母粉末の質量濃度が５ｇ／Ｌ、塩化ナトリウムの
質量濃度が１０ｇ／Ｌであり、残りは水であり、さらに６個の異なる減菌済コニカルフラ
スコに異なる濃度の目標抗生物質または目標抗生物質誘導体を加え、６個の異なる減菌済
コニカルフラスコ中の目標抗生物質または目標抗生物質誘導体の濃度はそれぞれ０．０１
μｇ／Ｌ、０．１μｇ／Ｌ、１μｇ／Ｌ、１０μｇ／Ｌ、１００μｇ／Ｌであり、２８℃
の室温条件下で１４日間振とう反応させて目標溶液を得る。
Example 8
This example differs from Example 1 in the following points:
S4-1, Environmental microorganism extraction: 50 parts by weight of the target watershed water sample was filtered through a filter membrane, the pore size of the filter membrane was 0.2 μm, and after filtration, the filter membrane was cut into pieces with an area of 15 mm2 , and the pieces were into a container and add a phosphate buffer solution of 1.2 times the fragment volume.In the phosphate buffer solution, the mass concentration of the NaCl solution is 10 g/L, and the mass concentration of the KCl solution is 0.25 g/L.
L, the mass concentration of the Na 2 HPO 4 solution is 1.6 g/L, and the mass concentration of the KH 2 PO 4 solution is 0.
3 g/L, the remainder is water, the pH of the phosphate buffer solution is 7.4, and after adding 2.5 times the fragment volume of glass beads to the container and shaking on a vortex for 15 min. , filter and remove the filter membrane and glass beads to obtain and prepare the bacterial solution supernatant,
S4-2, Antibiotic gradient concentration culture: Add the bacterial solution supernatant obtained in step S4-1 to five sterilized conical flasks, and add 2.5 parts by weight of bacteria to each sterilized conical flask. Three slides were placed in each sterilized conical flask containing the solution supernatant to simulate sediment biofilm formation, and 0.2 parts by weight of LB medium was added.
The mass concentration of tryptone is 10 g/L, the mass concentration of yeast powder is 5 g/L, the mass concentration of sodium chloride is 10 g/L, the remainder is water, and six different sterilized conical flasks have different concentrations. of target antibiotic or target antibiotic derivative were added, and the concentration of target antibiotic or target antibiotic derivative in six different sterile conical flasks was 0.01, respectively.
μg/L, 0.1 μg/L, 1 μg/L, 10 μg/L, 100 μg/L, 28°C
The target solution is obtained by performing a shaking reaction under room temperature conditions for 14 days.
実施例９
本実施例は、実施例１とは以下の点で異なり：
Ｓ４-１、環境微生物抽出：５０重量部の目標流域水試料をフィルター膜で濾過し、フィ
ルター膜の孔径は０．３μｍであり、濾過後、フィルター膜を面積２８ｍｍ２の断片に切
断し、断片を容器に入れ、断片体積の１．５倍のリン酸塩緩衝溶液を加え、リン酸塩緩衝
溶液では、ＮａＣｌ溶液の質量濃度が１０ｇ／Ｌ、ＫＣｌ溶液の質量濃度が０．２５ｇ／
Ｌ、Ｎａ２ＨＰＯ４溶液の質量濃度が１．６ｇ／Ｌ、ＫＨ２ＰＯ４溶液の質量濃度が０．
３ｇ／Ｌであり、残りは水であり、リン酸塩緩衝溶液のｐＨは７．４であり、さらに、容
器に断片体積の３倍のガラスビーズを添加し、ボルテックスで２０ｍｉｎ振とうした後、
フィルター膜とガラスビーズを濾過・除去して、細菌溶液上澄み液を得て用意し、
Ｓ４-２、抗生物質勾配濃度培養：ステップＳ４-１で得られた細菌溶液上澄み液をそれぞ
れ６個の減菌済コニカルフラスコに加え、各減菌済コニカルフラスコには３重量部の細菌
溶液上澄み液が含まれ、各減菌済コニカルフラスコに、堆積物のバイオフィルム形成を模
擬するためのスライドを３枚入れ、０．５重量部のＬＢ培地を加え、ＬＢ培地では、トリ
プトンの質量濃度が１０ｇ／Ｌ、酵母粉末の質量濃度が５ｇ／Ｌ、塩化ナトリウムの質量
濃度が１０ｇ／Ｌであり、残りは水であり、さらに６個の異なる減菌済コニカルフラスコ
に異なる濃度の目標抗生物質または目標抗生物質誘導体を加え、６個の異なる減菌済コニ
カルフラスコ中の目標抗生物質または目標抗生物質誘導体の濃度はそれぞれ０．０１μｇ
／Ｌ、０．１μｇ／Ｌ、１μｇ／Ｌ、１０μｇ／Ｌ、１００μｇ／Ｌであり、３３℃の室
温条件下で１４日間振とう反応させて目標溶液を得る。
Example 9
This example differs from Example 1 in the following points:
S4-1, Environmental microorganism extraction: 50 parts by weight of the target watershed water sample was filtered through a filter membrane, the pore size of the filter membrane was 0.3 μm, and after filtration, the filter membrane was cut into pieces with an area of 28 mm2 , and the pieces were into a container and add a phosphate buffer solution of 1.5 times the fragment volume.In the phosphate buffer solution, the mass concentration of the NaCl solution is 10 g/L and the mass concentration of the KCl solution is 0.25 g/L.
L, the mass concentration of the Na 2 HPO 4 solution is 1.6 g/L, and the mass concentration of the KH 2 PO 4 solution is 0.
3 g/L, the remainder is water, the pH of the phosphate buffer solution is 7.4, and after adding 3 times the fragment volume of glass beads to the container and shaking on a vortex for 20 min,
Filter and remove the filter membrane and glass beads to obtain and prepare the bacterial solution supernatant,
S4-2, Antibiotic gradient concentration culture: Add the bacterial solution supernatant obtained in step S4-1 to six sterilized conical flasks, and add 3 parts by weight of the bacterial solution supernatant to each sterilized conical flask. In each sterilized conical flask, 0.5 parts of LB medium was added, and the mass concentration of tryptone was 10 g/L, the mass concentration of yeast powder is 5 g/L, the mass concentration of sodium chloride is 10 g/L, the remainder is water, and six different sterilized conical flasks are charged with different concentrations of the target antibiotic or Target antibiotic derivative was added, and the concentration of target antibiotic or target antibiotic derivative in six different sterile conical flasks was 0.01 μg each.
/L, 0.1 μg/L, 1 μg/L, 10 μg/L, and 100 μg/L, and the target solution is obtained by shaking and reacting at room temperature at 33° C. for 14 days.
実験例
実施例１中の方法のパラメータを例にとると、目標抗生物質はテトラサイクリン、得られ
た目標抗生物質の濃度と選択性係数Ｓの対応関係図は図２に示され、この目標抗生物質は
目標溶液ＤＮＡの存在量から算出した耐性遺伝子の相対存在量の平均値Ｐを選択し、具体
的なパラメータは表１に示される。
Experimental Example Taking the parameters of the method in Example 1 as an example, the target antibiotic is tetracycline, and the correspondence diagram between the concentration of the target antibiotic and the selectivity coefficient S is shown in Figure 2. The average value P of the relative abundance of resistance genes calculated from the abundance of target solution DNA was selected, and the specific parameters are shown in Table 1.
Claims (6)
ステップＳ１、環境監視：
ステップＳ１-１：目標流域における抗生物質の生産、使用および排出の背景調査を行
い、生態リスク評価目標抗生物質の監視リストを確立し、目標抗生物質の環境監視を行い
、前記目標流域は河川流域、海域および水機能領域を含み、
ステップＳ１-２：目標流域における目標抗生物質誘導体を監視し、目標抗生物質の副
産物反応や生物変換が発生した場合、目標抗生物質誘導体監視リストを確立し、目標抗生
物質誘導体の環境監視を行う必要があり、
ステップＳ２、抗生物質初期スクリーニング：ステップＳ１-１で得られた目標流域の目
標抗生物質およびステップＳ１-２で得られた目標流域の目標抗生物質誘導体を初期スク
リーニングし、ステップＳ２-１～Ｓ２-２のいずれかを満たす場合、ステップＳ３微生物
薬品耐性評価に移行して継続的に評価し、それ以外の場合、評価を終了し、
ステップＳ２-１、オクタノール／水分配係数判定：目標抗生物質または目標抗生物質
誘導体のオクタノール／水分配係数Ｋｏｗを求め、取得したオクタノール／水分配係数Ｋ
ｏｗを１０の底を持つ対数値とし、ｌｇ Ｋｏｗ≧３．５の場合、この目標抗生物質また
は目標抗生物質誘導体が強脂溶性と判定し、ステップＳ３微生物薬品耐性評価に移行して
継続的に評価し、
ステップＳ２-２、抗生物質環境濃度判定：目標抗生物質または目標抗生物質誘導体の
環境濃度ＭＥＣを測定し、環境濃度ＭＥＣ≧１０の場合、この目標抗生物質または目標抗
生物質誘導体が連続的な入力源を有すると判定し、ステップＳ３微生物薬品耐性評価に移
行して継続的に評価する必要があり、
ステップＳ３、微生物薬品耐性評価：薬品耐性リスク指数ＲＱＲを用いて目標流域中の微
生物群集の薬品耐性リスクを特徴付け、以下の式に示すように：
ＲＱＲ＝ＭＥＣ／ＰＮＥＣＲ、
式において、ＭＥＣはステップＳ２-２で測定された目標抗生物質または目標抗生物質誘
導体の環境濃度であり、ＰＮＥＣＲは目標流域中の微生物群集の予測された薬品耐性無効
濃度であり、以下の式に示すように：
ＰＮＥＣＲ＝ＭＩＣ／ＡＦ、
式において、ＭＩＣは目標流域中の微生物群集の最小発育阻止濃度であり、ＡＦは１０の
値を取る無次元評価要因であり、ＥＵＣＡＳＴデータベースから目標流域中の微生物群集
の最小発育阻止濃度ＭＩＣを決定し、目標流域中の微生物群集の予測された薬品耐性無効
濃度ＰＮＥＣＲを取得し、前記微生物群集は細菌、真菌または放線菌属を含み、
目標流域中の微生物群集の薬品耐性リスクレベルは、ＲＱＲ値の大きさに応じて、ＲＱＲ
＜０．０１でリスクなし、０．０１＜ＲＱＲ＜０．１で低リスク、０．１＜ＲＱＲ＜１で
中リスク、ＲＱＲ＞１で高リスクという４クラスに分けられ、ＲＱＲ＞０．１のとき、目
標流域中の微生物群集の薬品耐性リスクレベルが高いと見なされ、ステップＳ４高レベル
評価に移行して継続的に評価し、それ以外の場合、評価を終了し、
ステップＳ４、高レベル評価：
ステップＳ４-１、環境微生物抽出：５０重量部の目標流域水試料をフィルター膜で濾
過し、前記フィルター膜の孔径は０．２～０．３μｍであり、濾過後、フィルター膜を面
積１５～２８ｍｍ２の断片に切断し、断片を容器に入れ、断片体積の１．２～１．５倍の
リン酸塩緩衝溶液を加え、容器に断片体積の２．５～３倍のガラスビーズを添加し、ボル
テックスで１５～２０ｍｉｎ振とうした後、フィルター膜とガラスビーズを濾過・除去し
て、細菌溶液上澄み液を得て用意し、
ステップＳ４-２、抗生物質勾配濃度培養：ステップＳ４-１で得られた細菌溶液の上澄
み液をそれぞれ５～６個の減菌済コニカルフラスコに加え、各減菌済コニカルフラスコに
は２．５～３重量部の細菌溶液上澄み液が含まれ、各減菌済コニカルフラスコに、堆積物
のバイオフィルム形成を模擬するためのスライドを３枚入れ、０．２～０．５重量部のＬ
Ｂ培地を加え、５～６個の異なる減菌済コニカルフラスコに異なる濃度の目標抗生物質ま
たは目標抗生物質誘導体を加え、５～６個の異なる減菌済コニカルフラスコ中の目標抗生
物質または目標抗生物質誘導体濃度はそれぞれ０、０．０１μｇ／Ｌ、０．１μｇ／Ｌ、
１μｇ／Ｌ、１０μｇ／Ｌ、１００μｇ／Ｌであり、２８～３３℃の室温条件下で１４日
間振とう反応させ、目標溶液を得、
ステップＳ４-３、ＤＮＡ抽出：ステップＳ４-２で得られた目標溶液から目標溶液ＤＮ
Ａを抽出する同時、減菌済コニカルフラスコ中のスライド上の細菌をリン酸塩緩衝溶液に
収集して細菌ＤＮＡを抽出し、
ステップＳ４-４、耐性遺伝子測定：目標抗生物質または目標抗生物質誘導体に対応す
る耐性遺伝子を測定し、目標溶液ＤＮＡまたは細菌ＤＮＡの存在量を測定して耐性遺伝子
の相対存在量の平均値Ｐを算出する同時に、ステップＳ４前の耐性遺伝子の相対存在量の
平均値Ｐ０を算出し、選択性係数Ｓを算出し、以下の式に示すように：
Ｓ＝Ｉｎ（Ｐ／Ｐ０）
ステップＳ４-５、モデルフィッティング：抗生物質濃度Ｃを横座標とし、目標溶液Ｄ
ＮＡまたは細菌ＤＮＡに対応する選択性係数Ｓを縦座標として、ｌｏｇｉｓｔｉｃモデル
を用いてフィッティングし、Ｓ＝０のとき、対応の抗生物質濃度ＣはＭＳＣ値であり、目
標溶液ＤＮＡまたは細菌ＤＮＡのＭＳＣ値を比較し、小さいＭＳＣ値を管理基準として選
択し、このＭＳＣ値は、抗生物質濃度Ｃより高い抗生物質条件下で薬品耐性遺伝子を有す
る微生物が濃縮され、その結果、微生物群集中の薬品耐性遺伝子の相対存在量が増加する
、
ことを特徴とする水環境中の残留抗生物質に対するマルチレベルのリスク評価方法。 including steps S1 to S4,
Step S1, environmental monitoring:
Step S1-1: Conduct a background investigation on the production, use and discharge of antibiotics in the target basin, establish an ecological risk assessment monitoring list for the target antibiotics, perform environmental monitoring of the target antibiotics, and the target basin is a river basin. , including marine and water functional areas;
Step S1-2: Monitor the target antibiotic derivative in the target watershed, and if by-product reactions or biotransformations of the target antibiotic occur, establish a target antibiotic derivative monitoring list and perform environmental monitoring of the target antibiotic derivative. There is,
Step S2, initial antibiotic screening: Initial screening of the target antibiotic in the target area obtained in step S1-1 and the target antibiotic derivative in the target area obtained in step S1-2 is carried out, and steps S2-1 to S2- If either of 2 is satisfied, proceed to step S3 Microbial drug resistance evaluation and continuously evaluate; otherwise, end the evaluation,
Step S2-1, determination of octanol/water partition coefficient: determine the octanol/water partition coefficient Kow of the target antibiotic or target antibiotic derivative, and obtain the obtained octanol/water partition coefficient K
If ow is a logarithmic value with a base of 10, and lg Kow≧3.5, the target antibiotic or target antibiotic derivative is determined to be strongly lipophilic, and the process proceeds to Step S3, microbial drug resistance evaluation, where it is continuously evaluated. Evaluate,
Step S2-2, antibiotic environmental concentration determination: measure the environmental concentration MEC of the target antibiotic or target antibiotic derivative, and if the environmental concentration MEC≧10, this target antibiotic or target antibiotic derivative is a continuous input source. It is necessary to proceed to step S3 microbial drug resistance evaluation and continuously evaluate.
Step S3, Microbial drug resistance evaluation: The drug resistance risk index RQ R is used to characterize the drug resistance risk of the microbial community in the target watershed, as shown in the following formula:
RQ R =MEC/PNEC R ,
In the formula, MEC is the environmental concentration of the target antibiotic or target antibiotic derivative measured in step S2-2, PNEC R is the predicted drug resistance null concentration of the microbial community in the target watershed, and the following formula As shown:
PNEC R = MIC/AF,
In the formula, MIC is the minimum inhibitory concentration of the microbial community in the target watershed, AF is a dimensionless evaluation factor that takes a value of 10, and the minimum inhibitory concentration MIC of the microbial community in the target watershed is determined from the EUCAST database. obtain a predicted drug resistance null concentration PNECR of a microbial community in a target watershed, the microbial community comprising bacteria, fungi, or the genus Actinobacteria;
The drug resistance risk level of the microbial community in the target watershed is determined by the RQ R value.
RQ R is divided into four classes: no risk if <0.01, low risk if 0.01<RQ R <0.1, medium risk if 0.1<RQ R <1, and high risk if RQ R >1. >0.1, the drug resistance risk level of the microbial community in the target watershed is considered to be high, and the process moves to step S4 high level evaluation to continuously evaluate; otherwise, the evaluation is terminated;
Step S4, high level evaluation:
Step S4-1, environmental microorganism extraction: 50 parts by weight of the target watershed water sample is filtered with a filter membrane, the pore size of the filter membrane is 0.2~0.3μm, and after filtration, the filter membrane is divided into an area of 15~28mm. Cut into 2 pieces, put the pieces in a container, add 1.2 to 1.5 times the volume of the phosphate buffer solution, and add 2.5 to 3 times the volume of glass beads to the container. After shaking with a vortex for 15 to 20 minutes, filter and remove the filter membrane and glass beads to obtain and prepare a bacterial solution supernatant.
Step S4-2, Antibiotic gradient concentration culture: Add the supernatant of the bacterial solution obtained in step S4-1 to 5 to 6 sterilized conical flasks, and add 2. Contains ~3 parts by weight of bacterial solution supernatant, each sterile conical flask contains three slides to simulate sediment biofilm formation, and 0.2 to 0.5 parts by weight of L
Add B medium and add different concentrations of target antibiotic or target antibiotic derivative to 5-6 different sterilized conical flasks, add target antibiotic or target antibiotic derivative in 5-6 different sterilized conical flasks. The substance derivative concentrations are 0, 0.01 μg/L, 0.1 μg/L, respectively.
1 μg/L, 10 μg/L, 100 μg/L, and a shaking reaction was performed for 14 days under room temperature conditions of 28 to 33°C to obtain the target solution.
Step S4-3, DNA extraction: target solution DN from the target solution obtained in step S4-2
At the same time as extracting A, collect the bacteria on the slide in a sterile conical flask into a phosphate buffer solution to extract bacterial DNA;
Step S4-4, resistance gene measurement: measure the resistance gene corresponding to the target antibiotic or target antibiotic derivative, measure the abundance of the target solution DNA or bacterial DNA, and calculate the average value P of the relative abundance of the resistance gene. At the same time as calculating, the average value P0 of the relative abundance of resistance genes before step S4 is calculated, and the selectivity coefficient S is calculated, as shown in the following formula:
S=In(P/P 0 )
Step S4-5, model fitting: antibiotic concentration C is the abscissa, target solution D
A logistic model is used to fit the selectivity coefficient S corresponding to NA or bacterial DNA as the ordinate, and when S=0, the corresponding antibiotic concentration C is the MSC value, and the MSC of the target solution DNA or bacterial DNA The values were compared and the smaller MSC value was selected as the control standard. This MSC value indicates that microorganisms with drug resistance genes are concentrated under antibiotic conditions higher than the antibiotic concentration The relative abundance of genes increases,
A multi-level risk assessment method for antibiotic residues in the aquatic environment.
ーとモデル予測方法を組み合わせて目標抗生物質または目標抗生物質誘導体の半減期ｔ１
／２を求め、環境残留性レベルは半減期ｔ１／２値の大きさに応じて、ｔ１／２＜６０ｄ
で非残留性、６０ｄ＜ｔ１／２＜１８０ｄで残留性、ｔ１／２＞１８０ｄで高残留性とい
う３クラスに分けられ、ｔ１／２＞６０の場合、目標抗生物質または目標抗生物質誘導体
が環境残留性を有すると判定し、すなわち、目標流域中の微生物群集の薬品耐性リスクＲ
ＱＲの値にかかわらず、ステップＳ４に移行して継続的に評価する必要がある、ことを特
徴とする請求項１に記載の方法。 The step S3 further includes the following steps: environmental persistence evaluation: combining the database query and model prediction method to determine the half-life t 1 of the target antibiotic or target antibiotic derivative;
/2 , and the environmental persistence level is determined as t 1/2 <60d depending on the half-life t 1/2 value.
It is divided into three classes: non-residual at 60d < t 1/2 < 180 d, persistent at t 1/2 > 180 d, and when t 1/2 > 60, the target antibiotic or target antibiotic It is determined that the derivative has environmental persistence, i.e., the drug resistance risk R of the microbial community in the target watershed.
The method according to claim 1, characterized in that regardless of the value of QR , it is necessary to proceed to step S4 and continuously evaluate.
０ｇ／Ｌ、ＫＣｌ溶液の質量濃度が０．２５ｇ／Ｌ、Ｎａ２ＨＰＯ４溶液の質量濃度が１
．６ｇ／Ｌ、ＫＨ２ＰＯ４溶液の質量濃度が０．３ｇ／Ｌであり、残りは水であり、リン
酸塩緩衝溶液のｐＨは７．４である、ことを特徴とする請求項１に記載の方法。 In the phosphate buffer solution in steps S4-1 and S4-3, the mass concentration of the NaCl solution is 1.
0 g/L, the mass concentration of KCl solution is 0.25 g/L, the mass concentration of Na 2 HPO 4 solution is 1
．． 6 g/L, the mass concentration of the KH 2 PO 4 solution is 0.3 g/L, the remainder is water, and the pH of the phosphate buffer solution is 7.4. Method described.
の質量濃度が５ｇ／Ｌ、塩化ナトリウムの質量濃度が１０ｇ／Ｌであり、残りは水である
、ことを特徴とする請求項１に記載の方法。 In the LB medium in step S4-1, the mass concentration of tryptone is 10 g/L, the mass concentration of yeast powder is 5 g/L, the mass concentration of sodium chloride is 10 g/L, and the remainder is water. A method according to claim 1, characterized in that:
に記載の方法。 Claim 1, wherein in step S4-4, 2 to 20 types of resistance genes are obtained.
The method described in.
るマルチレベルのリスク評価システムであって、
目標流域内の目標抗生物質および目標抗生物質誘導体の種類および含有量を監視するため
に使用され、まず情報処理装置において目標流域内の目標抗生物質監視リスト、目標抗生
物質誘導体監視リストを作成し、その後目標抗生物質および目標抗生物質誘導体の監視を
実行する環境監視システムと、
情報処理装置上で実際に検出した目標抗生物質、目標抗生物質誘導体と、環境監視システ
ムにより建築された目標抗生物質監視リスト、目標抗生物質誘導体監視リストとを比較し
、実際に検出した目標抗生物質、目標抗生物質誘導体が環境監視システムにより建築され
た目標抗生物質監視リスト、目標抗生物質誘導体監視リストに属するかどうかを判定する
初期スクリーニングシステムと、
情報処理装置上で、前記初期スクリーニングシステムの判定結果がＹｅｓである目標抗生
物質、目標抗生物質誘導体に対して微生物薬品耐性評価を行う微生物薬品耐性評価システ
ムと、
情報処理装置上で微生物薬品耐性リスクレベルの高い目標抗生物質、目標抗生物質誘導体
をさらに評価して、制限または禁止する必要のある生態学的リスクの高い抗生物質種類を
特定する高レベル評価システムと、を備える、ことを特徴とする水環境中の残留抗生物質
に対するマルチレベルのリスク評価システム。 A multi-level risk assessment system for antibiotic residues in an aquatic environment, used in the method according to any one of claims 1 to 5, comprising:
It is used to monitor the types and contents of target antibiotics and target antibiotic derivatives in the target watershed, and first, a target antibiotic monitoring list and a target antibiotic derivative monitoring list in the target watershed are created in the information processing device, an environmental monitoring system that subsequently performs monitoring of the target antibiotic and target antibiotic derivative;
The target antibiotics and target antibiotic derivatives actually detected on the information processing device are compared with the target antibiotic monitoring list and target antibiotic derivative monitoring list constructed by the environmental monitoring system, and the target antibiotics actually detected are determined. , an initial screening system for determining whether the target antibiotic derivative belongs to a target antibiotic derivative monitoring list, a target antibiotic derivative monitoring list established by an environmental monitoring system;
a microbial drug resistance evaluation system that performs microbial drug resistance evaluation on a target antibiotic or target antibiotic derivative for which the determination result of the initial screening system is Yes on an information processing device;
A high-level evaluation system that further evaluates target antibiotics and target antibiotic derivatives with a high risk level of microbial drug resistance on an information processing device to identify types of antibiotics with a high ecological risk that need to be restricted or prohibited. A multi-level risk assessment system for antibiotic residues in a water environment, comprising:
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| JP2006034950A (en) * | 2004-05-27 | 2006-02-09 | Sankyo Co Ltd | ACTIVE DECOMPOSITION AND REMOVAL METHOD OF beta-LACTAM COMPOUND USING NUCLEOPHILIC REAGENT |
| JP2019521682A (en) * | 2016-07-04 | 2019-08-08 | アリファックス ソチエタ レスポンサビリタ リミタータＡｌｉｆａｘ Ｓ．Ｒ．Ｌ． | Integrated device for performing diagnostic analysis |
| JP2020198878A (en) * | 2019-06-06 | 2020-12-17 | 南京▲農業▼大学 | Test paper strips and methods for detecting tetracycline antibiotics in water using biosensors |
| JP7009688B1 (en) * | 2021-09-15 | 2022-01-26 | 南京大学 | Sensor array and detection device consisting of LOMF for detecting antibiotics |
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| US20230392183A1 (en) | 2023-12-07 |
| CN115062933B (en) | 2023-04-18 |
| JP7408041B2 (en) | 2024-01-05 |
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| CN115062933A (en) | 2022-09-16 |
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